The efforts of the prior art to express structural information for the production of eukaryotic cell proteins in transformed bacteria vary widely in their approach. One class of prior art cloning vehicles, which is represented by those described in European Patent Publication No. 1929 (published on May 16, 1979) and in Goeddel, D. V., et al., Nature 281, 544-548 (1979), is preferably constructed in accordance with a scheme in which the structural gene for the desired polypeptide (e.g., somatostatin) is inserted (together with a translation termination codon) at a restriction endonuclease recognition site located within the E. coli .beta.-galactosidase structural gene, bringing the expression of the desired polypeptide under the control of the .beta.-galactosidase or "lac" promoter. Since some of the codons for the .beta.-galactosidase enzyme are thereby positioned to be translated in advance of the structural gene of the desired product, expression of this fused DNA sequence in an E. coli transformant is said to result in a precursor protein comprising a somatostatin (or other) polypeptide preceded by a superfluous protein fragment consisting of either a sequence of several amino acid residues derived from the .beta.-galactosidase structural gene or virtually the entire .beta.-galactosidase amino acid sequence, depending upon which insertion site is chosen.
Another effort involves use of the lac UV5 promoter in a bacteriophage vehicle, .lambda., or in a plasmid vehicle pBR322 (Charnay, P., et al., Nucleic Acids Res. 5, 4479-4494 [1978]). In both cases, each vector allows the fusion of a cloned gene to the lacZ gene in a different phase relative to the translation initiation codon of the lacZ gene. Used in combination, these vectors are said to allow translation of a cloned gene in any one of the three coding phases.
The fact that this prior art scheme contemplates expression of the conjugate precursor protein under the control of the lac promoter gives rise to certain disadvantages. The machinery for gene expresion associated with the control region of the .beta.-galactosidase gene is not as efficient as the machinery for gene expression associated with certain other bacterial genes. Accordingly, the yield of the expression product is expected to range from approximately 100,000 molecules per host cell up to a maximum of about 200,000 molecules per host cell. Furthermore, the efficiency of this system may be further reduced because the mRNA molecule transcribed from the DNA of these prior art cloning vehicles is unnecessarily long (extending in some cases far "upstream" and in other cases far "downstream" of the desired polypeptide through the remainder of the .beta.-galactosidase structural gene), and may lack certain features, called "secondary structures" or "stem-and-loop structures," which are believed to confer greater stability upon the mRNA molecule, thereby increasing its availability for ribosomal translation. Finally, the fused precursor-protein expression product accumulates inside the transformant cell, perhaps interfering with normal cellular activities and certainly requiring cell disruption in the harvesting process.
A second class of prior art cloning vehicles, represented by those described in Seeburg, P. H., et al., Nature 276, 795-798 (1978), involves the expression of similar "hybrid" proteins. In this scheme, the structural gene for the desired polypeptide is inserted within the structural gene for the .beta.-lactamase (or penicillinase) enzyme of E. coli. Although this protein is normally synthesized with a short leader sequence (also called a "signal peptide"), which is located at the amino terminus of the protein and is thought to direct secretion of the protein across the cytoplasmic membrane, the expected fused precursor-protein expression product of such a recombinant vehicle (containing the DNA sequence for rat growth hormone) in an E. coli transformant was not actually detected outside the cell, despite a reasonable expectation that a .beta.-lactamase-rat growth hormone conjugate would be secreted into the periplasmic space.
In any event, harvest of this product is expected to yield only approximately 24,000 molecules per host cell, and other workers using this scheme for expression of rat insulin have estimated an even smaller yield, on the order of 100 molecules per host cell (see VillaKomaroff, L., et al., Proc. Natl. Acad. Sci. U.S.A. 75, 3727-3731 [1978]). It is believed that the relatively modest recovery of the desired product which might be obtained with such cloning vehicles is due to the relative weakness of the E. coli .beta.-lactamase promoter, under the control of which the fussion product is expressed, as compared with the promoters of certain other components of the E. coli genome.
A third class of prior art cloning vehicles is represented by those described in Tacon, W., et al., Molec. gen. Genet. 177, 427-428 (1980) and in Hallewell, R. A. and Emtage, S., Gene 9, 27-47 (1980), wherein the use of the E. coli tryptophan gene is described. ln the former case, three plasmids are constructed from pBR322 which contain the E. coli tryptophan promoter-operator, as well as nucleotides specifying the leader sequence and first seven amino acids of the trpE gene. These plasmids have a Hind III cloning site situated downstream from the translation initiation codon (ATG) of the trpE gene, with the cloning site in each plasmid differing in its translation phase relative to the initiation codon. In the latter case, the tryptophan promoter-operator, the trpE gene and 15% of the trpD gene are used for the expression of foreign genes.
In summary, the prior art has failed to develop cloning vehicles which are suited for the efficient expression of prokaryotic and eukaryotic gene products in bacterial transformants, and which at the same time utilize functional fragments such as the 3'-untranslated region or the transcription termination site of any gene for expression of an exogenous DNA insert fragment. Furthermore, the prior art has failed to utilize more than one promoter in a single expression plasmid to control the production of the desired protein. It is therefore the principal object of the present invention to provide a new class of plasmid cloning vehicles incorporating not only the promoter and 5'-untranslated region, but also the 3'-untranslated region and the transcription termination site, derived from a particular class of bacterial genes, and incorporating a second promoter fragment as well.